Recent interest has centered on the role of adenosine 5',5'''-P1-P4- tetraphosphate (Ap4A) in cell division. This molecule is markedly increased in proliferating cells, binds to DNA polymerase and stimulates DNA synthesis in vivo. Also, tRNA-4-Lys has been implicated in cell division and appears to be required for cell to transverse G1 and enter S phase. These two cell responses have recently been linkedxby our demonstration that tRNA-4-Lys preferentially induces the synthesis of Ap-4-A by lysyl-tRNA synthetase in vitro. Comparisons will be made between the 6 eucaryotic tRNALys isoacceptors and correlated to aminoacylation and Ap4A synthetic activity. The presence or absence of two modified nucleosides results in a 7-fold difference in Ap4A synthesis, but no difference in aminoacylation activity when assayed with purified lysyl-tRNA synthetase under the same conditions. Purified tRNA-4-Lys will be chemically modified at specific sites to test the structural requirements for synthetase recognition in both aminoacylation and Ap4A synthesis. The mechanism of Ap4A synthesis in the presence of 2.5 MuM zinc and tRNA will be determined by reaction kinetics, binding studies and specific exchange reactions, and compared to the mechanism at 80 MuM zinc which does not require the presence of tRNA. Comparisons will also be made between the activity of free and aggregated synthetase.